NobleGen Biosciences was founded based upon the groundbreaking work of Dr. Amit Meller's lab at Boston University in which they demonstrated the proof of concept for a unique, nanopore based method for DNA sequencing by nanopore induced photon emission (SNIPE), which utilizes optical detection rather than the more ubiquitous electrical detection. The process is now more generally referred to as Opti-pore" sequencing. The Meller approach is superior to other nanopore approaches under development in that: i) the high density nanopore array, potentially comprising 10,000 to >100,000 nanopores, does not require development of technology for individually addressable nanopores, ii) the readout is performed on a synthetic representation mitigating known issues with reading genomic sequence variations being passed through a nanopore, and iii) the system is modular in that biochemistry and readout are separable. Critical for the Opti-pore process is the proprietary biochemical DNA conversion approach, Circular DNA Conversion (CDC). The fundamental proof of CDC has been shown in a stylized version wherein encoding of a single base with high accuracy for all 4 bases was demonstrated. In this SBIR grant I propose two distinct aims, which when brought together, will become a critical component in the Opti-pore process with the potential to enable DNA sequencing at an unprecedented scale in terms of speed (>2 10^6 bases/s) and extremely low cost. My first aim is to develop a synthetic probe library design to include several critical features: primer binding sites, restriction binding sites one of which is a Type IIs enzyme, and molecular beacon 4 color base coding, to enable cyclical processing and 4 color readout from the synthetic representation of 100 - 1000's of bases from the source genomic material. Our focus on each aspect of development is taking basic concepts demonstrated in the Meller lab and then screening a variety of enzymes and reaction conditions with the goal of maximizing specificity and yield for each step of the process. Our second aim is to develop and optimize the various individual steps of Circular DNA conversion (CDC) cycle wherein each cycle is comprised of: hybridizations, ligations, and RE cutting. We plan on achieving this second aim via internal research and collaboration with vendors who have demonstrated their interest and ability to provide commercially available enzymes (ligases and Type IIs REs) of suitable quality for single molecule biochemical process. As proof of the process success, the synthetic representation will be sequenced using the industry standard Sanger method to validate the process produces a high accuracy representation. Additionally, the synthetic representations produced during this optimization process will be verified at various stages by actual Opti-pore sequencing in the laboratory of Dr. A Meller. Successfully completing these two aims, I will have achieved demonstrating cyclical processing for conversion of the sample genome into the synthetic representations for the commercialization of the Opti-pore process: a radically new, cost- effective DNA sequencing platform, capable of long read lengths, high speed, and high accuracy. This is expected to have a wide-ranging impact on both basic and applied biomedical research and personalized healthcare.